B 13Galactosyltransferase

a1,3-Galactosyltransferase (a1,3-GalT, EC 2.4.1.151) has recently attracted much attention because it catalyzes the transfer of Gal from UDP-Gal to 3-OH of the Gal residue in Gal£1 — 4GlcNAc-R to form Gala1 — 3Gal£1 — 4GlcNAc epitopes on glycoproteins and glycolipids (Fig. 4) [65-67]. This is the major xenoactive antigen that is responsible for hyperacute rejection in xenotransplantation [68,69]. a1,3-Galactosyltransferases from porcine and bovine tissues and recombinant bovine a1,3-galactosyltransferase have all been used for preparative synthesis, the latter for gram-scale reactions [66,70]. Recombinant porcine a1,3-GalT is now commercially available in unit quantities, and a fusion protein comprising UDP-Gal epimerase and a1,3-GalT has been engineered for large-scale synthesis [71].

a1,3-Galactosyltransferase transfers 3-deoxy-, 4-deoxy-, and 6-deoxy-Gal from the corresponding donor at very low rates (<2%), while UDP-2-deoxy-Gal is a better

Figure 4 The reaction catalyzed by a!,3-galactosyltransferase.

substrate than UDP-Gal [72] (Fig. 5). However, unlike the case of 01,4-GalT, UDP-Glc, GalNAc, GlcNAc, and glucuronic acid are not transferred by a1,3-GalT [73].

Acceptors other than Gal^l ^ 4GlcNAc are utilized by a1,3-GalT, including Gal^l ^ 3GlcNAc and Gal^l ^ 4Glc [67,74], and acceptors immobilized on polymers [71]. Modifications on 4-OH of Gal in acceptors give inactive compounds, suggesting that this is a key polar group essential for binding to a1,3-GalT [67]. The N-acetyl group in acceptors can be replaced with azido, succinimido, and others with a large number of acyl groups [67,75] (Fig. 5). However, replacement of the 2-NHAc with an amino group abolishes activity [73]. As shown in Figure 5, analogs with modifications on 6-OH of GlcNAc, or 2-OH and 6-OH of the terminal Gal residue are substrates, while only deoxygenation of the 3-OH of GlcNAc residue is tolerated [67]. The enzyme can catalyze the transfer of a Gal residue to a very hindered tertiary alcohol acceptor where the carbon-bonded hydrogen at the glycosylation site is replaced with a methyl group, demonstrating that glycosyltransferases can overcome inherent steric limitations to produce oligosaccharide analogs that cannot be easily prepared by chemical methods [76].

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